Previously, we (BF and BP) made full length "infectious" cDNA clones from a virulent dengue type 1 virus (DEN1 WP) and from a live-attenuated DEN1 vaccine candidate, which had been adapted to grow in dog kidney cells (DEN1 PDK20). Transfection of cells with RNA transcribed in vitro from these clones produces a dengue infection. Recovered viruses behave like the corresponding parent DEN1 in growth curves, and the recovered DEN1 PDK20 has the same small plaque phenotype as its parent. Work has begun to map the mutations responsible for this small plaque phenotype, by making chimeras between the WP and PDK20 infectious clones. Also, in human clinical trials the PDK20 virus is slightly too reactogenic, yet a PDK26 virus is overattenuated. We plan to introduce further attenuating mutations into the PDK20 clone in an effort to recover a correctly attenuated vaccine strain, and at first we will use subsets of the mutations found in PDK26 which are not in PDK20. Another project currently underway in collaboration with B Puri is to make an infectious clone of a DEN type 4 PDK-adapted vaccine candidate. So far, we have succeeded in making a full-length cDNA clone; the infectivity of transcripts is currently being investigated. In collaboration with E Kelly, we have made another infectious clone, of a PDK adapted DEN2 vaccine candidate. The sequence of the clone was compared to the population average sequence of the parent vaccine candidate, and several differences are being repaired. Next on the agenda is to compare this virus to its parent in tissue culture cells and in small animals. The ultimate plan for this clone is to manufacture a candidate vaccine under GMP (to be done at WRAIR by K. Eckels) and to compare the transcript- derived virus to its uncloned parent in human trials. Finally, last year we (BF and CZ) completed an infectious clone of the Army's candidate live attenuated Japanese encephalitis virus (JEV) vaccine, a vero cell adapted version of a PDK cells derivative of the Chinese JEV live vaccine strain SA14-14-2. The phenotype of virus recovered from this clone is being investigated in growth curves in cells and in mouse immunogenicity studies.